Essential oils are gaining significant importance in food, cosmetic and pharmaceutical sectors due to their diverse biological effects (Bakkali et al., 2008). They are commonly obtained from different parts of plants, such as leaves, flowers and stems and roots (Ali et al., 2015). The genus Alpinia (Zingiberaceae family), found abundantly in tropical Asia, is known for its high content of volatile oils and terpenoids (Padalia et al., 2010). Recent studies have highlighted the antioxidant, antihypertensive and antimicrobial properties of essential oils from this genus (Zhang et al., 2016).
       
Traditional methods like hydro-distillation (HD) and steam distillation are widely used for essential oil extraction (Cassel et al., 2009; Tongnuanchan et al., 2014). However, these methods are often limited by long processing times and high energy consumption (Rostagno et al., 2013). Furthermore, the high temperatures can lead to the degradation of unsaturated compounds and the loss of volatile active ingredients, thereby reducing the quality and value of the essential oil.
       
Optimized essential oil extraction methods demonstrate notable efficiency. For instance, the optimization of essential oil extraction from Cinnamomum camphora and Rumex Crispus using microwave-assisted hydro-distillation (MAHD) has been reported (Hundie et al., 2023; Shang et al., 2020). Furthermore, other studies from the field of Agricultural Science and comparisons between MAHD and traditional distillation methods highlight the potential advantages of MAHD (Thimmappa et al., 2023; Moradi et al., 2018).
       
To address these limitations, microwave-assisted extraction (MAE) has emerged as a superior alternative (Nagahata et al., 2019). MAE is a rapid and efficient technique that utilizes microwave energy to heat the plant material, leading to faster cell rupture and more effective extraction of compounds (Dao et al., 2020). This method not only improves the electrical energy conversion to thermal energy but also accelerates chemical processes (Chanthai et al., 2012).
       
This study utilized MAHD with the aim of enhancing the yield of essential oil extraction from the leaves of Alpinia blepharocalyx K. Schum. The research aimed to determine the optimal conditions for maximizing both essential oil yield and DPPH radical scavenging activity. By employing response surface methodology (RSM) with a central composite design (CCD), we systematically focused on how three specific parameters-microwave power, liquid-to-material ratio and extraction time affected the process (Bezerra et al., 2008).

Material and reagents
 
Alpinia blepharocalyx leaves were gathered from Ky Son District, Nghe An Province, Vietnam. The samples were cleaned to remove pests and old parts, then washed and dried at 40^oC. The dried leaves were pulverized and passed through a 1.0 mm mesh screen. The treated samples were kept refrigerated at a temperature range of 0^oC to 4^oC for later experiments.
 
Microwave-assisted hydro-distillation
 
The microwave-assisted hydro-distillation (MAHD) system consists of a microwave oven and a Clevenger apparatus (Fig 1). First, 50 g of dried A. blepharocalyx leaf samples were transferred to a distillation flask, followed by the addition of deionized water. The mixture was then heated with microwave radiation. After a set period, the essential oil fraction was collected in a separate bottle. To remove any remaining water, anhydrous sodium sulfate was added to the essential oil, followed by centrifugation.


     
The yield of essential oil is calculated via the ratio of the the dried essential oil mass (DMO) to dried leaf sample mass (DML) as shown in the Equation 1:

   
DPPH radical scavenging assay (RSA)
 
A. blepharocalyx leaf extract was evaluated using the the DPPH radical scavenging test. This method measures the capacity of the extract to scavenge 1,1-diphenyl-2-picrylhydrazyl radicals. The experimental procedure was performed by combining 0.2 mL of the sample extract and 3.8 mL of a DPPH ethanolic solution (5.9 mg DPPH in 100 mL ethanol). The mixture was stirred for 1 minute and then left to stand for 30 minutes at ambient temperature and protected from light. Absorbance readings were taken at 517 nm using an Agilent 8453 UV-visible Spectrophotometer.
     
The experiment was replicated with triplicate with all determination’s samples. The DPPH (%) was calculated according to Equation 2:

Abs_sample, Abs_control are noted for the absorbance of A. blepharocalyx leaf extract and the absorbance of blank control, respectively.
 
Experimental design
 
To optimize the extraction conditions, using three-factor response surface method analysis using a central composite design (CCD) with parameters (Microwave power (W), Liquid-to material ratio (mL/g) and extraction time (min)). In which, each experiment using the total amount of water is 100 g. Table 1 displays the coded independent variables for the RSM design.


       
In this research, using the program Design-Expert® to the evaluated effects of the extraction parameters. The quadratic polynomial model of response variable was ditermined as Equation 3:

Where,
Yi = Predicted response.
β₀, β_i, β_ii = Regression coefficient.
x_i, x_j = Different independent variables.
X_i and X_j = Coded independent variables.
 
Determine the chemical composition of A. blepharocalyx leaves essential oil by Gas Chromatography-Mass Spectrometry (GC-MS)
 
The essential oil was chemically characterized by a Trace GC Ultra - ITQ900 GC-MS system (Thermo, USA). The oven temperature was programmed as follows an initial hold at 60^oC for 2 min, a ramp to 200^oC at 4^oC/min, followed by another ramp to 360^oC at 10^oC/min, with a final 10 min hold for column clean-up. The injector temperature was 280^oC. A 1.0 µL sample, diluted in n-hexane (1/50, v/v), was injected in split mode (split ratio: 1/15) (Yim et al., 2011).

Regression model analysis
 
Using the central composite design with 20 actual experiments with 3 factors to optimize the yield of A. blepharocalyx leaves essential oil and the DPPH radical scavenging activity values of extract fluids, are shown in Table 2.


       
From the experimental results and linear regression analysis of 20 experiments, regression equations were proposed of two responses (essential oil yield and DPPH):
 

Y₁ = 3.28 + 0.24X₁ + 0.024X₂ + 0.29X₃ - 0.071X₁X₂ - 0.034X₁X₃ - 0.056X₂X₃ - 0.13X₁² - 4.36X₂² - 0.46X₃²                                                                                         

Y₂ = 87.68 - 3.9X₁ - 0.18X₂ - 0.52X₃ - 01.67X₁X₂ + 4.82X₁X₃ + 0.39X₂X₃ - 1.71X₁² - 0.26X₂² - 0.14X₃²                                                                                 

       
The experiments were conducted using a central composite design with 20 runs to optimize the essential oil yield (Y₁) and DPPH radical scavenging activity (Y₂). The analysis of variance (ANOVA) for the quadratic regression model (Table 3) confirmed that the models were highly significant for both responses (p<0.0001) (Rezzoug et al., 2005). The coefficients of determination (R²) were 0.9596 for essential oil yield and 0.9933 for DPPH activity, indicating that the models could explain 95.96% and 99.33% of the variability in the responses, respectively (Chanthai et al., 2012).


 
Response surface analysis
 
The three-dimensional plots of the response surface (Fig 2 and Fig 3) clearly illustrate the effects and interactions of the independent variables on the responses.




 
Essential oil yield (Y₁)
 
• Effect of microwave power and liquid-to-material ratio: At a constant extraction time of 40 min, the oil yield increased with higher microwave power. The yield also increased as the liquid-to-material ratio increased from 5:1 to 7:1 mL/g but decreased with further increases, suggesting an optimal point around this ratio.
 
• Influence of microwave power and extraction time: With a fixed liquid-to-material ratio of 7:1 mL/g, the oil yield was positively correlated with an increase in both microwave power and extraction time, reaching a peak at the central levels.

• The increase in essential oil yield with higher microwave power (from 500 W to 700 W) can be attributed to the efficient heating mechanism of microwaves. Microwave radiation directly interacts with polar molecules, primarily the intracellular water within the plant matrix. This interaction causes rapid, localized superheating, leading to a dramatic increase in internal pressure that ruptures the cell walls and oil glands. Consequently, the essential oil is released more rapidly and completely into the surrounding solvent, thus enhancing the extraction yield.
 
DPPH radical scavenging activity (Y₂)
 
• Effect of microwave power: DPPH activity showed an inverse relationship with microwave power, decreasing as the power increased. This could be due to the degradation of some antioxidant compounds at higher energy levels.
 
• Influence of liquid-to-material ratio and extraction time: DPPH activity reached a maximum at a liquid-to-material ratio of approximately 7.5:1 mL/g, then decreased. The activity also decreased as the extraction time increased, suggesting that prolonged exposure to microwave radiation may reduce the antioxidant capacity of the extract.
 
• Interestingly, an inverse relationship was observed between microwave power and DPPH radical scavenging activity. While higher power enhances yield, the associated increase in temperature likely promotes the degradation of thermolabile antioxidant compounds. Components such as certain oxygenated monoterpenes or phenolic compounds, which are known contributors to antioxidant capacity, may have been partially decomposed or volatilized at higher energy levels, leading to a reduction in the overall radical scavenging ability of the extracted oil.
 
Optimization and model verification
 
The optimal conditions predicted by the RSM model were a microwave power of 602.49 W, a liquid-to-material ratio of 6.99:1 mL/g and an extraction time of 45.44 min. These predicted values were highly consistent with the experimental results obtained from a validation experiment conducted under slightly modified, practical conditions: A microwave power of 600 W, a liquid-to-material ratio of 7.0:1 mL/g and an extraction time of 45 min (Table 4). The experimental values for essential oil yield (3.35±0.02%) and DPPH activity (87.05±0.05%) were very close to the predicted values, validating the model’s reliability.


 
Chemical profile of essential oils
 
The GC-MS analysis identified 39 chemical components in the essential oil extracted under optimal conditions (Table 5). The major compounds were β-Pinen (20.9%), 1,8-Cineol (20.8%) and α-Pinen (13.2%).


       
The chemical profile of A. blepharocalyx essential oil in our study was dominated by β-Pinene (20.9%), 1,8-Cineol (20.8%) and α-Pinene (13.2%). This composition shows some similarities to a previous report on the same species from Vietnam by Hung et al. (2018), who also identified 1,8-Cineol and α-Pinene as major constituents, although in different proportions. However, our finding of β-Pinene as a primary component differs from their report, suggesting that factors such as geographical location, harvest season or the extraction method (MAHD vs. traditional hydrodistillation) could signi-ficantly influence the chemical chemotype of the essential oil.

This study successfully demonstrated the effectiveness of MAHD for extracting essential oil from Alpinia blepharocalyx leaves. By using response surface methodology, we were able to optimize the extraction process and identify the most favorable conditions for maximizing both essential oil yield and antioxidant activity. An optimal microwave power of 600 W was achieved, a liquid-to-material ratio of 7.0:1 mL/g and an extraction time of 45 min. These findings provide a robust scientific foundation for the efficient and high-quality production of essential oil from A. blepharocalyx on a larger scale.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.